The cardiovascular system is a major determinant of the body's oxidative metabolism and aerobic capacity; thus, successful cardiovascular aging is critical to maintenance of an active, independent lifestyle in elderly individuals. Aging of the cardiovascular system results in progressive endothelial dysfunction that is associated with increased risk for cardiovascular disease and a loss of skeletal muscle performance. The endothelial dysfunction present in skeletal muscle resistance arteries of aged rats results primarily from reduced nitric oxide (NO) signaling;however, the effects of age on cellular mechanisms that regulate NO signaling remain to be determined. Recent evidence indicates that the availability of tetrahydrobiopterin (BH4), a necessary cofactor in NO production, is decreased in skeletal muscle resistance arteries of aged rats. Inadequate availability of BH4 can result in biochemical uncoupling of endothelial nitric oxide synthase (eNOS) and reduced synthesis of NO. Furthermore, uncoupled eNOS produces superoxide anion (021 a reactive oxygen species which can limit NO signaling and contribute to cellular damage. The overarching goals of this proposal are 1) to determine whether aging results in uncoupling of eNOS and reduction of bioavailable NO in skeletal muscle resistance arteries, and 2) to determine whether interventional strategies, including aerobic exercise training, can reverse age-related deficiencies in BH4 availability and uncoupling of eNOS, increasing NO bioavailability in skeletal muscle resistance arteries. In Aim 1 NO and O2- will be measured directly to determine whether aging produces uncoupling of eNOS in resistance arteries from rat soleus muscle leading to reduced NO bioavailability, and increased production of cytotoxic O2-. In Aim 2, endothelium-specific adenoviral gene transfer will be used to determine whether restoration of BH4 availability by overexpression of GTP cyclohydrolase (GTPCH) reverses age-related uncoupling of eNOS, increasing NO bioavailability and reducing O2- in soleus muscle resistance arteries. shRNAi knockdown of GTPCH will also be used to reduce BH4 availability and promote eNOS uncoupling in soleus muscle resistance arteries. The purpose of Aim 3 is to determine whether chronic aerobic exercise training reduces oxidant stress and increases GTPCH expression thereby increasing BH4 availability and reversing age-related uncoupling of eNOS and NO bioavailability in resistance arteries from rat soleus muscle. These studies will increase our understanding of mechanisms that contribute to age-related endothelial dysfunction in resistance vasculature of skeletal muscle and indicate whether specific interventions designed to improve BH4 availability can ameliorate endothelial dysfunction in old age